Experiments were carried out using male Sprague-Dawley rats weighing between 180 and 210 g. The animals were maintained in a temperature-controlled room (23 ± 1°C) with a 12/12 hour light-dark cycle. All procedures involving the use of the animals were approved by the Institutional Animal Care and Use Committee of the School of Dentistry, Kyungpook National University, and were carried out in accordance with the ethical guidelines for the investigation of experimental pain in conscious animals proposed by the International Association for the Study of Pain.
Development of animal model by LPA injection into the trigeminal nerve root
Surgical procedures were performed under pentobarbital sodium (40 mg/kg, i.p.) anesthesia. Anesthetized rats were mounted on a stereotaxic frame (Model 1404; David Kopf Instruments, Tujunga, CA) for LPA injection into the left trigeminal nerve root. A 3 μL volume of LPA (1 nmol) was injected into the trigeminal nerve root through a glass micropipette (tip diameter 50 μm) connected to a 10 μL Hamilton syringe (31 gauge) for 10 sec. The site of injection was 7.2 mm posterior to the bregma, 2.8 mm lateral from the midline, and 9.6 mm ventral from the surface of the skull. Ten minutes later, the glass micropipette was withdrawn, the wound was sutured, and a local anesthetic ointment was applied. After each experiment, the rats were intracardially perfused with 100 ml of heparinized normal saline followed by 500 ml of freshly prepared fixative solution under deep anesthesia with sodium pentobarbital (80 mg/kg, i.p.). After perfusion, the LPA injection site was identified using a stereoscope. Only data obtained from rats with injection sites clearly within the trigeminal nerve root were used in the behavioral analysis. LPA was purchased from Biomol and dissolved in 0.25% methanol in saline.
General procedures for behavioral testing
Rats were randomly assigned to one of three groups. In the LPA-treated group (n = 16), the animals received an LPA injection into the trigeminal nerve root. In the control group, the rats received a vehicle injection (n = 8). Behavioral responses were also examined in a naïve group (n = 7) that did not receive any surgery or treatments. All behavioral tests were conducted between 0700 and 1800 hours. Rats were tested 3 days before and at 3, 7, 10, 14, 17, 21, 24, 30, 40, 55, 70, 85, 100, 130 and 160 days after the LPA injection. We measured the body weights of the animals after behavioral monitoring and returned them to their cages. All behavioral responses were measured by an experimenter in a blind fashion.
Evaluation of orofacial mechanical allodynia
To conduct behavioral observations, each rat was placed in a customized observation cage which was then placed in a darkened and noise-free room. The animals were then acclimated for at least 30 min. Withdrawal behavior, produced by 10 successive trials of constant air-puff pressure (4 sec duration, 10 sec intervals), was then examined in freely moving rats, as described previously [31, 49, 50]. The intensity and intervals of the air-puff pressure were controlled with a pneumatic pump module (BH2 system; Harvard Apparatus, Holliston, MA). The air-puffs were applied through a 26-gauge metal tube (length, 10 cm) located 1 cm from the skin at a 90° angle. Thresholds were determined through the air-puff pressure at which each rat responded in 50% of the trials. The cut-off pressure for the air-puffs was 40 psi. The naïve rats did not respond to a pressure of less than 40 psi. A significant decrease in air-puff thresholds compared with the pre-operative values was defined as mechanical allodynia.
Evaluation of orofacial mechanical hyperalgesia
Mechanical hyperalgesia was assayed using a pin-prick test following the same strict rule of habituation and testing environments for von Frey testing, as described previously [31, 50]. Briefly, the rats were left to adapt to the observation cage for 30 min prior to the actual stimulation. During this period, an experimenter reached into the cage every 30 sec to touch the walls of the cage with a plastic rod, similar to those used to induce pain, as previously described . After the rats became accustomed to the reaching movements, a series of mechanical stimulations were conducted. A blunt acupuncture needle (Needle No. 3, Serine, Japan) was applied until the needle was slightly bent (the skin was dimpled but not penetrated). The rat response to the stimuli was scored on an ordinal scale [31, 49, 50] as follows: no response, 0; detection, 1; detection and withdrawal, 2; detection, withdrawal, and escape or attacking movements, 3; as for response 3, but with prolonged facial grooming (> 3 strokes), 4.
Evaluation of orofacial thermal hyperalgesia
Each rat was placed in a customized cylinder-type acrylic rodent restrainer (height 40 mm-60 mm, length 70-120 mm) for the evaluation of thermal hyperalgesia. The cages had a hole in the top so that the head could receive thermal stimulation and produce a head withdrawal action. Each cage was placed in a darkened and noise-free room and the animals were habituated for at least 30 min prior to the start of the experiment. After radiant heat was applied, the head withdrawal time was determined as described in previous studies [31, 52]. The application of heat stimuli was performed using an infrared thermal stimulator (Infrared Diode Laser, LVI-808-10; LVI tech, Seoul, Korea). The power and current of the thermal stimulator were adjusted to 11 W and 18.1 A, respectively. This intensity of the thermal stimuli produced stable latencies of approximately 12 sec for a 10 cm distance from the heat source to the vibrissa pad. Each rat received two stimuli and the inter-stimulus interval for each trial was at least 5 min. A cut-off time of 20 seconds was used in these experiments to prevent possible tissue damage.
Under deep anesthesia with sodium pentobarbital (80 mg/kg, i.p.), rats (n = 6) were intracardially perfused with 100 ml of heparinized normal saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) on postoperative day 14 or 160. After perfusion, the LPA injection site was identified. The trigeminal nerve root was then removed, postfixed in the same fixatives overnight, and then embedded in paraffin and sectioned (4 μm thick) for light microscopy. Sections were stained with luxol fast blue, which identifies the myelin sheaths of the axons. The histology of each section was examined under a light microscope.
Evaluation of antinociceptive and neuroprotective effects of progesterone
To investigate the antinociceptive effects of progesterone, we administered this hormone subcutaneously in LPA-treated animals. Progesterone (8, 16 mg/kg/day) was injected beginning on operative day for five consecutive days. This concentration of progesterone was shown previously to recover behavioral performance in rats after medial frontal cortical contusions . After these daily treatments, changes in the air-puff thresholds were measured at 30, 60, 120, 180 min, and 24 hours. We also tested for any delayed anti-allodynic effects of progesterone after the fifth treatment. To investigate neuroprotective effects of progesterone following the final administration of progesterone at 16 mg/kg/day, changes in P0 or PMP 22 immunoreactivity were examined. Progesterone was purchased by Sigma and dissolved in 200 μl sesame oil.
After the fifth treatment with progesterone, some of the rats (n = 5 per group) were perfused through the ascending aorta with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PB. The trigeminal nerve roots were then dissected out, postfixed in the same fixative at 4°C overnight, and then replaced with 30% sucrose in 0.1 M PB overnight. Transverse frozen sections (free-floating, 18 μm) were performed by a cryostat and processed for immunohistochemistry. All sections were blocked with 5% goat serum in PBS containing 0.2% Triton X-100 for 1 h at room temperature. Subsequently, they were incubated overnight at 4°C with rabbit polyclonal anti-P0 (1:500; Abcam, Cambridge, UK) or rabbit polyclonal anti-PMP22 (1:500; Abcam). The sections were then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibody (Vector Laboratories, Burlingame, CA) for 2 h at room temperature. The sections were next covered with DAB solution (Vector Laboratories) for 10 min and observed under a light microscope (Axioplan; Zeiss, Oberkochen, Germany).
Rats (n = 5 per group) were sacrificed by decapitation after their fifth treatment with progesterone. The trigeminal nerve root including the injection site was immediately removed and quickly frozen in liquid nitrogen. Samples were sonicated with Biorupture (Cosmo Bio., Tokyo, Japan) in a lysis buffer containing protease and a phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL). Protein concentrations in the samples were measured using a fluorometer (Invitrogen, Carlsbad, CA). For western blotting, total proteins (30 μg) were separated in a 4-12% gradient NuPAGE Novex Bis-Tris gel (Invitrogen) and transferred onto a PVDF membrane using an iBlot Dry blotting system (Invitrogen). The membranes were then blocked with 5% non-fat milk in TBS with 0.1% Tween 20 for 1 h at room temperature and then incubated with P0 (1:2000, Abcam) or PMP 22 (1:2000, Abcam) at 4°C overnight. The blots were subsequently incubated with goat anti-rabbit horseradish peroxidase for 1 h at room temperature. Membranes were developed using the SuperSignal West Femto substrate (Pierce, Rockford, IL), and exposed to X-ray film. We used the ImageJ analysis system (NIH, Bethesda, MD) to quantify specific bands.
Changes in motor performance after the subcutaneous administration of progesterone were measured using a rotarod (Ugo Basile, Comerio-Varese, Italy), as described previously [54, 55]. The rotarod speed was set at 12 rpm with a maximum time spent on the rod set at 180 sec. The rats received two or three training trials on two separate days prior to testing for acclimatization. On the testing day, the resting response was examined. After the subcutaneous administration of progesterone (16 mg/kg/day), the time course of the motor performance was examined.
Differences between groups were compared using analysis of repeated measures analysis of variance (ANOVA) followed by LSD post hoc analysis. Comparisons between two means were performed using the Student's T-test. In all statistical comparisons, P < 0.05 was used as the criterion for statistical significance. All data are presented as the mean ± SEM.